T-box genes form an ancient family of putative transcriptional regulators characterized by a region of homology to the DNA-binding domain of the murine Brachyury (T) gene product. This T-box domain is conserved from Caenorhabditis elegans to human, and mutations in T-box genes have been associated with developmental defects in Drosophila, zebrafish, mice, and humans. Here we report the identification of three novel murine T-box genes and an investigation of their evolutionary relationship to previously known family members by studying the genomic structure of the T-box. All T-box genes from nematodes to humans possess a characteristic central intron that presumably was inherited from a common ancestral precursor. Two additional intron positions are also conserved with the exception of two nematode T-box genes. Subsequent intron insertions, potential deletions, and/or intron sliding formed a structural basis for the divergence into distinct subfamilies and a substrate for length variations of the T-box domain. In mice, the 11 T-box genes known to date can be grouped into seven subfamilies. Genes assigned to the same subfamily by genomic structure show related expression patterns. We propose a model for the phylogenetic relationships within the gene family that provides a rationale for classifying new T-box genes and facilitates interspecific comparisons.